1. Field of the Invention
The present invention relates to a method for synthesis of nucleic acids, especially to a method for synthesis of nucleic acids by means of a polymerase chain reaction (hereinafter abbreviated as a PCR).
2. Description of the Related Art
A PCR method is a procedure capable of amplifying an intended DNA fragment as much as several hundred thousand-fold by repeating a process comprised of dissociation of a DNA strand into single strands, binding of primers with sandwiching a particular region of the DNA strand, and a DNA synthesis reaction by a DNA polymerase. The PCR method is described in Japanese Laid-open Patent Publication No.S61-274697 which is an invention by Mullis et al.
A PCR procedure can be used as a highly sensitive method for analyzing nucleic acids in various samples. For animals, the PCR procedure is used for searching for genes that cause an infectious disease, a hereditary disease or the like, searching for therapeutics utilizing those genes, diagnosis, monitoring, or the like. The PCR procedure is also suited to DNA typing tests for a transplantation, a paternity test, medical treatments based on an individual genetic information, and the like. For these purposes, a peripheral blood is often selected as a test object. In addition, for plants, the PCR procedure is used for searching for useful genes, monitoring of plants in which these genes are transduced, or the like. For these purposes, a leaf is often selected as a test object.
One drawback of the PCR procedure is that the reaction is inhibited by pigments, proteins, saccharides, or unknown contaminants. Namely, many DNA polymerases including Taq DNA polymerase derived from Thermus aquaticus, a typical thermostable DNA polymerase, are widely known to allow the PCR to be inhibited potently by even a trace amount of living body-derived contaminants existing in the PCR reaction solution.
Therefore, the PCR procedure requires a process in which a cell(s), a fungus (fungi), a bacterium (bacteria), a virus(es) or the like (hereinafter referred to as a nucleic acid inclusion body) are isolated from a subject and then nucleic acids are extracted from the nucleic acid inclusion body prior to a DNA amplification. Such process has conventionally been a procedure in which the nucleic acid inclusion body is decomposed using an enzyme, a surfactant, a chaotropic agent, or the like, and then nucleic acids are extracted from the decomposed product of the nucleic acid inclusion body using, for example, phenol or phenol/chloroform.
Recently, an ion-exchange resin, a glass filter, glass beads, a reagent having an effect of agglutinating proteins, or the like is used in the step of the nucleic acid extraction.
It is difficult, however, to completely remove impurities by purifying nucleic acids in a sample using these procedures, and furthermore, an amount of nucleic acids in a sample recovered by these purification procedures often varies among experiments. For these reasons, a subsequent nucleic acid synthesis may sometimes be unsuccessful, especially when a content of the intended nucleic acid in the sample is low. In addition, these purification procedures involve complicated manipulations and are time-consuming, and there is a high opportunity for contamination during the procedures.
Therefore, a simpler, more convenient and effective method of a sample pretreatment is desired in order to solve these problems.